Embryonic and other pluripotent stem cells have great potential in therapy. Such cells can be directed to differentiate into specific cell types and offer the possibility of a renewable source of replacement cells and tissues, for example, for use in regenerative medicine to repair tissues which have been damaged by disease or injury.
However, the use of embryonic stem cells in medicine is limited due to the significant ethical concerns associated with the use of embryos. The Yamanaka Lab2 and Thomson Lab3 demonstrated that human fibroblasts can be reprogrammed by the transient overexpression of a small number of genes into induced pluripotent stem cells (IPSCs) which functionally and phenotypically resemble embryonic stem cells (ESCs). Thus, pluripotent stem cells can be obtained without the need for the destruction of embryos.
Some IPSCs, like hESC, express Oct-4 and other cell surface markers, such as Tra-1-60/81 and SSEA-3/4. However, IPSCs are not identical to ESCs, as shown by a slower doubling time11, differences in the global gene-expression patterns2,3 and DNA methylation status2. It remains unknown whether nuclear reprogramming is complete10 and thus whether IPSCs follow a similar pathway to hESCs during differentiation.
This important breakthrough raises the possibility that cellular therapies using patient-specific input cells may be a reality in the future. Unlike hESC where there are ethical concerns and possible issues of immune rejection, IPSCs can be generated from a donor, reprogrammed, differentiated to the appropriate cell type and transplanted back into the donor.
Prior to the publication of reports that IPSCs had been successfully generated from human cells, we described the generation of a panel of monoclonal antibodies (mAbs) against surface antigens on undifferentiated hESCs1 in WO 2007/102787, the contents of which is hereby incorporated in this application by reference. These mAbs showed strong reactivity against undifferentiated, but not differentiated (embryoid bodies), hESC lines.
The mAbs did not cross react with mouse fibroblasts, and showed weak to no reactivity against human embryonal carcinoma cells. Thus these mAbs exhibited very high specificity binding to hESCs, and this binding was lost as the hESCs differentiated. The monoclonal antibody, mAb 84, is an IgM which specifically binds and kills undifferentiated human embryonic stem cells (hESC) (Tan, 200929). mAb 84 induced cell death of undifferentiated, but not differentiated hESC within 30 min of incubation, and immunoprecipitation of the mAb-antigen complex revealed that the antigen is podocalyxin-like protein-1 (PODXL). Importantly, the absence of tumour formation is observed when hESC were treated with mAb 84 prior to transplantation into SCID mice. This earlier data indicates that mAb84 may be useful in eliminating residual undifferentiated hESC from differentiated cell populations for clinical applications.
In addition to generating antibodies which were cytotoxic against undifferentiated hESCs, such as mAb84, in WO 2007/102787 we also produced mAbs which were non-cytotoxic against undifferentiated hESCs. One notable non-cytotoxic antibody was mAb 85.
Furthermore, in WO/2010/033084, the contents of which are hereby incorporated by reference, we reported the discovery of mAbs that bind to and characterize IPSCs. We reported that mAb 84 was cytotoxic against IPSCs, and mAb85 was non-cytotoxic against IPSCs.
Although undifferentiated stem cells may be used in cell therapy, it is considered to be beneficial to use cells which have started to differentiate, or are differentiated. Methods of encouraging stem cells to differentiate into particular cell lineages are well known in the art. Once the differentiation process has started or proceeded, it is beneficial to remove or destroy undifferentiated hESCs in a sample which may otherwise form undesirable teratomas. Teratomas typically contain a mixture of differentiated or partly differentiated cell types. Despite the potential of IPSC therapy, the problem of teratoma formation by residual IPSC after differentiation remains and needs to be addressed.
Thus, mAb 84 can potentially be used for separation and removal of residual undifferentiated hESC or undifferentiated IPSC from differentiated cell populations.
Thus, it can be seen that it is useful to identify, isolate or separate undifferentiated pluripotent stem cells (since they can be used themselves in therapy or can be encouraged to differentiate into a particular cell lineage which can be used in therapy). It is also useful to remove or destroy undifferentiated pluripotent stem cells from a mixture of cells where some of the cells have started to differentiate, or are differentiated, since these differentiated cells are useful in therapy.
It can also be seen that there is a need to identify further antibodies, in addition to mAb84, which are specifically cytotoxic against both hESCs and IPSCs. Such antibodies could then be useful in methods for separation and removal of residual undifferentiated hESC or undifferentiated IPSC from differentiated cell populations, as described above.
The present invention arose as a result of ongoing studies by the present inventors into the areas described above. The inventors were in possession of cytotoxic and non-cytotoxic antibody molecules which were known to bind PODXL. They also knew that PODXL was a glycosylated protein. The inventors went on to investigate the importance of glycosylation for the binding of antibody molecules to PODXL. The inventors approached this by testing cytotoxic and non-cytotoxic antibody molecules in a glycan array against a range of different glycans. The inventors found that cytotoxic and non-cytotoxic antibody molecules bound to a common sequence of saccharide residues within glycans. Surprisingly, the inventors also found that there was a significant difference in the binding of cytotoxic and non-cytotoxic antibody molecules to this common sequence. Hence the inventors found that candidate cytotoxic antibody molecules can be selected based on their ability to bind glycans containing the common sequence.